| Hucho genus belongs to Salmonidae, Salmoniformes. There are 5 species in the world:Hucho hucho, distributed in Danube basin, Europe; Hucho taimen, widely distributed in the northern Eurasia; Hucho bleekeri, distributed in Yangtze River drainage, China; Hucho ishikawae, distributed in the upper reaches of the Yalu River, China and North Korea;Hucho perryi, distributed in Sakhalin Island and Hokkaido and the Far East of Japan. They have important academic value to research biogeography, fish phyletic evolution and paleoecology.Chuanshan taimen (Hucho bleekeri), also known as Sichuan taimen, is the only species endemic to China among the Hucho genus. Its distribution area was large before 1960s. In recent years, the natural resources experienced serious decline due to many factors such as destruction of the ecological environment, over fishing, and so on. It has disappeared without a trace in most of its usual distribution areas in Shanxi and Sichuan provinces, and facing the threat of extinction. The 19 adults of Hucho bleekeri found in Taibai River in 2012 were the largest group in recent 20 years, and this was a rediscovery in Taibai River since this species disappeared in this area 15 years ago. The protection plan of this species was set up. The protection of genetic diversty is an important factor in the research of conservation genetics. Molecular biology techniques are effective methods to protect species from extinction. In this study, we studied the population genetics of H.bleekeri, developed microsatellite markers, assessed the genetic diversity and population structure,established paternity test technology,and researched the introgression ofH.bleekeri and Brachymystax lenok tsinlingensis. Furthermore, we researched the molecular phylogeny of this species. Specific findings are as follows:1 Development of 27 novel cross-species microsatellite markers for the endangered Hucho bleekeri using next-generation sequencing technologyWe developed 27 novel cross-species microsatellite markers for the endangered Hucho bleekeri using next-generation sequencing technology. The number of alleles varied from 2 to 8 for each primer set. The observed and expected heterozygosity ranged from 0.150 to 0.906 and 0.508 to 0.845, respectively. The polymorphic information content ranged from 0.371 to 0.808. These microsatellite loci should be useful to study population genetics,paternity identification, and speciation and adaptive evolution of this lineage.2 Conservation genetics assessment and phylogenetic relationships of critically endangered Hucho bleekeri from Taibai River using mitochondrial DNA fragments and microsatellite markersMitochondrial DNA fragments: we collected 43 wild individuals of H. bleekeri, and used control region and CYTB gene fragment to assess the genetic diversity of this population. There were 10 polymorphic sites in the control region but no singleton variable site, and two polymorphic sites in the CYTB gene, with two singleton variable sites. Both the number of haplotypes (h) and the haplotype diversity (Hd) of the control region (12 haplotypes and Hd = 0.8208) were higher than those of the CYTB gene fragment (three haplotypes and Hd = 0.0941). The nucleotide diversity (Pi) of both markers was very low:0.00159 for control region and 0.00009 for the CYTB gene. The average numbers of nucleotide differences (k) were also low: 1.871 for the control and 0.095 for the CYTB gene.The consequence of network showed a relatively single structure of this population.Phylogenetic analysis showed no obvious differentiation in this population.Microsatellite markers: we chose 15 primers with high polymorphism and marked them with 2 kinds of fluorescence in the forward primer respectively (FAM or HEX). We found a total of 77 alleles in the H. bleekeri population, ranging from 2 (Hb110-16) to 13 (Hbl8-163) per locus. The calculated Ho varied from 0.04651 to 1.00000, with a mean of 0.59719, whereas He ranged from 0.09111 to 0.67387, with a mean of 0.44735. Most of the 15 microsatellite loci deviated significantly from HWE (P < 0.05), except Hbl2-2. The coefficient of inbreeding (Fis) ranged from 0.90487 to 0.49398, with a mean of 0.34033,and was not significant (P > 0.05). Significant (P < 0.05) linkage disequilibrium was not detected in any loci combinations. No obvious genetic differentiation was found in this population. And it suffered severe bottleneck effect recently (P < 0.05).The results of these two molecular markers all indicated that the genetic diversity of this wild population of H. bleekeri from Taibai River was very low.3 Genetic diversity assessment of first filial generation of breeding group and Establishment of paternity technology of Hucho bleekeriWe selected 14 highly polymorphic microsatellite primers from earlier study to estimate the genetic diversity of artificial breeding F1 population. The results showed that the number of microsatellite loci alleles (Na) ranged from 2 to 6, average number was 3; the observed heterozygosity (Ho) ranged from 0.069 to 1, and average value was 0.574; the expected heterozygosity (He) ranged from 0.067 to 0.696, average value was 0.432; the polymorphism information content (PIC) ranged from 0.064 to 0.644, average value was 0.339. The degree of genetic differentiation between two families (FST) was 0.02504,indicating that no significant genetic differentiation existed in the population. The single population inbreeding coefficient (Fis) was -0.33255, which showed that no obvious inbreeding existed in the population. Compared with the wild group, the parameters of genetic diversity of offspring group were significantly lower.We used 14 highly polymorphic microsatellite primers marked with fluorescent to construct the paternity test technology. Results showed that the combined non-exclusion probability (first parent) was more than 99%, the combined non-exclusion probability(parent pair) was more than 98%. All the offspring can correctly find their real parents.Results showed the 14 polymorphic microsatellite markers were suitable for paternity test of Hucho bleekeri.4 Research of introgression in Hucho bleekeri and Brachymystax lenok tsinlingensisWe analyzed the genetic characteristics of two species distributed in Taibai River,Qinling Mountains using mitochondrial DNA markers. And we made sequences alignment and clustering analysis between our data and published data of Hucho bleekeri from Make River, Qinghai Province and Brachymystax lenok from Heilongjiang drainage. Results showed that number of parsimony informative sites was 97, the coefficient of genetic differentiation was 0.06653, and the average number of nucleotide differences was 0.3476,nucleotide diversity was 0.08598, gene flow was 3.51. The fixed coefficient of the general population was 0.1080. No common haplotypes was detected in this two species.Phylogenetic trees showed that the group from two different groups. The results declared that there was no introgression in analysised DNA region between Hucho bleekeri and Brachymystax lenok tsinlingensis.5 Molecular phylogenetic position and differentiation years of Hucho bleekeriThe phylogenetic tree was contructed based on full-length of mitochondrial DNA and 13 protein-coding genes of most Salmonidae fishes. We speculated the divergence times of Hucho based on molecular clock with a constant evolutionary rate. Phylogenetic trees showed that H. bleekeri, H. taimen and H. hucho were in the same cluster, but branch of H.perryi and Salvelinus were close. Most posterior probability of nodes were more than 95%,showed this analysis had a high credibility.Therefore,it was inferred that H. perryi first separated from other taimens,with a divergence time of 10.4 Ma.H. bleekeri and H. hucho differentiated before 1.7?2.2 Ma,so as with H. taimen. H. hucho and H.taimen differentiated at 1.4?1.8 Ma. H.bleekeri collected from Makehe River and Taibai River differentiated at 1.3 ?1.7 Ma. And H.bleekeri and B. lenok tsinlingensis distributed in Qinling area isolated from species in north area at about 1.7 ?2.2 Ma. We speculated that these two species of Salmonidae diffused from north area and settled in this area in Pleistocene. |
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